Sharn Freier scored an early goal for Australia but reigning Olympic champions Canada soon equalised, and a late Evelyne Viens strike proved the winner in Marbella, Spain on Saturday.
Star Caitlin Foord was a late withdrawal for the match after feeling quad tightness during her warm-up, replaced in the starting lineup by Freier.
Sharn Freier capitalised on her late starting call up, scoring Australia's goal. (Lukas Coch/AAP PHOTOS)
She was one of several squad members deemed unavailable for the fixture as the Matildas focus on their Olympic campaign starting on July 25.
Kaitlyn Torpey and Steph Catley were ruled out earlier in the week with lower leg injuries but are on track to rejoin the squad at training prior to that opening match against Germany.
Goalkeeper Teagan Micah and midfielder Tameka Yallop were also ruled out as precautionary measures.
Coach Tony Gustavsson had warned of Canada's high press and that was soon evident with goalkeeper Mackenzie Arnold forced into a sharp save off Adriana Leon.
However, the Matildas struck first after Mary Fowler found space in Canada's half and played through a pinpoint ball to Freier, who beat a defender and slotted it past goalkeeper Kailen Sheridan.
Canada hit back late in the half after Cloe Lacasse's cross proved difficult for Arnold, opening the door for an onrushing Nichelle Prince to score.
Left-back Clare Wheeler gave Australia hope of regaining the lead just before half time but her pass across the face of goal could not meet Ellie Carpenter's back-post run.
Gustavsson made five substitutions at halftime with Charli Grant, Clare Polkinghorne, Emily van Egmond, Michelle Heyman and Courtney Nevin replacing Carpenter, Alanna Kennedy, Katrina Gorry, Hayley Raso and Cortnee Vine.
Polkinghorne's experience was on show as she made a vital block from a Jordyn Huitema shot just over 10 minutes into the half.
Viens eventually made the difference after a ball from Huitema found her in the box and she was able to rifle in a decisive finish, giving Canada the lead for the first time in the 85th minute.